scholarly journals Differential binding cell-SELEX method to identify cell-specific aptamers using high-throughput sequencing

2018 ◽  
Author(s):  
Karlis Pleiko ◽  
Liga Saulite ◽  
Vadims Parfejevs ◽  
Karlis Miculis ◽  
Egils Vjaters ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
Negative Selection ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Kidney Tissue ◽  
Live Cells ◽  
Aptamer Selection ◽  
Differential Binding ◽  
Binding Cell

ABSTRACTAptamers have in recent years emerged as a viable alternative to antibodies. High-throughput sequencing (HTS) has revolutionized aptamer research by increasing the number of reads from a few (using Sanger sequencing) to millions (using an HTS approach). Despite the availability and advantages of HTS compared to Sanger sequencing, there are only 50 aptamer HTS sequencing samples available on public databases. HTS data in aptamer research are primarily used to compare sequence enrichment between subsequent selection cycles. This approach does not take full advantage of HTS because the enrichment of sequences during selection can be due to inefficient negative selection when using live cells. Here, we present a differential binding cell-SELEX (systematic evolution of ligands by exponential enrichment) workflow that adapts theFASTAptamertoolbox and bioinformatics tooledgeR, which are primarily used for functional genomics, to achieve more informative metrics about the selection process. We propose a fast and practical high-throughput aptamer identification method to be used with the cell-SELEX technique to increase the aptamer selection rate against live cells. The feasibility of our approach is demonstrated by performing aptamer selection against a clear cell renal cell carcinoma (ccRCC) RCC-MF cell line using the RC-124 cell line from healthy kidney tissue for negative selection.

scholarly journals Differential binding cell-SELEX method to identify cell-specific aptamers using high-throughput sequencing

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Karlis Pleiko ◽  
Liga Saulite ◽  
Vadims Parfejevs ◽  
Karlis Miculis ◽  
Egils Vjaters ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Differential Binding ◽  
Binding Cell

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

scholarly journals Betanodavirus infection in Kuhlia rupestris and Ambassis marianus and isolation of betanodavirus from infected pond water

2020 ◽  
Vol 142 ◽  
pp. 1-11
Author(s):  
K Agnihotri ◽  
R Chong ◽  
D Underwood ◽  
C Kistler ◽  
M Hutchison
Keyword(s):  
Phylogenetic Analysis ◽  
Nucleotide Sequence ◽  
Cell Line ◽  
Environmental Samples ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Pond Water ◽  
Fish Cell ◽  
Nervous Necrosis Virus ◽  
Necrosis Virus

This is the first report of betanodavirus infection in 2 species of finfish, Kuhlia rupestris (jungle perch) and Ambassis marianus (estuary perchlet). This report also describes isolation of betanodavirus from infected pond water using the SSN-1 cell line. Histopathology of K. rupestris larvae revealed vacuolation in the eye and brain, which was confirmed using betanodavirus-specific immunohistochemistry. The eye and brain from A. marianus and betanodavirus isolated from pond water were confirmed using real-time PCR and Sanger sequencing. High throughput sequencing was used to obtain betanodavirus sequences from paraffin blocks containing infected K. rupestris. The phylogenetic analysis of betanodavirus RNA1 and RNA2 sequences from all 3 sources were associated with the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The RNA1 nucleotide sequence from jungle perch showed 100% identity with the betanodavirus water isolate and 99.37% identity with A. marianus. Furthermore, we have demonstrated the usefulness of combining recovery of viable virus from environmental samples through fish cell line culture with PCR testing as a means of validating the efficacy of chlorination to eradicate betanodavirus from the pond environment.

scholarly journals High-throughput Sequencing for Species Authentication and Contamination Detection of 63 Cell Lines

2021 ◽  
Author(s):  
Oliver Lung ◽  
Rebecca Candlish ◽  
Michelle Nebroski ◽  
Peter Kruckiewicz ◽  
Cody Buchanan ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Classical Swine Fever ◽  
Virus Genome ◽  
Cox1 Gene ◽  
Species Identity ◽  
Cell Line Authentication ◽  
Dna And Rna

Abstract Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency’s National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvβ6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

Sanger sequencing of mitochondrial hypervariable region of ancient samples for DNA authentication and screening before high-throughput sequencing

2021 ◽  
Vol 40 ◽  
pp. 103216
Author(s):  
Anna Šenovská ◽  
Eva Drozdová ◽  
Kristýna Brzobohatá ◽  
Eva Chocholová ◽  
Dana Fialová ◽  
...  
Keyword(s):  
High Throughput ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Hypervariable Region

scholarly journals Ighv Mutational Status By Deep Next Generation Sequencing Refines Ighv Sanger Sequencing Classification in Patients with Chronic Lymphocytic Leukaemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3028-3028
Author(s):  
Azahara Fuentes ◽  
Alicia Serrano ◽  
Blanca Ferrer Lores ◽  
Veronica Lendinez ◽  
Carolina Monzo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
High Throughput ◽  
Research Funding ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Magnetic Beads ◽  
Lymphocytic Leukaemia ◽  
Standard Technique ◽  
Mutational Status ◽  
Major Clone

Introduction: Determination of the mutational status of rearranged immunoglobulin heavy chain variable (IgHV) genes in patients with Chronic Lymphocytic Leukaemia (CLL), is considered one of the most important prognostic factors: patients with unmutated IgHV (UM; ≥98% of identity to the germline) genes have a more aggressive disease course and develop more frequently unfavourable genetic deletions or mutations than patients with mutated IgHV (M; ≤98%). Mutational status, is currently determined by Sanger sequencing (Sseq) that allows the analysis of the major clone, however, international guidelines recommend caution in assigning mutational status in cases with "Borderline" IgHV identity (97-97.9%), and cases with double rearrangements with discordant mutational status. Objective: Analyze and determine the mutational status of the IgHV locus by High-throughput sequencing (HTS), in a cohort of CLL patients (n=51) with unclassifiable Sseq results: borderline status (n=22); double rearrangements (n=27) with discordant mutational status (n=2). Methods: We included 51 DNA samples extracted from peripheral blood of patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2019 (median absolute lymphocytes 11.4x109/L [2,8-239,5x109/L]). Sseq amplification and analysis of IgHV rearrangements were performed on DNA conforming to the updated ERIC recommendations. In all the cases we were able to determinate the IGVH identity. To switch high-throughput sequencing to the clinical practice, we assessed the reliability of different library preparation methods to sequence IGH locus in patients with CLL. Amplification was performed using the Sequencing Multiplex Kit based on IGH FR (forward primers) and consensus JH (reverse primer) multiplex. PCR products were purified using Magsi-NGS Prep magnetic beads (Magnamedics Diagnostics), normalized and pooled to create a library for sequencing using a MiSeq equipment. To simplify and make automatic the analysis of the same we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements. Results: This approach led to the identification of a dominant clone IgHV in all cases (n=51). Instead, the percentage of identity calculated by HTS analysis varies in: - 15/22 borderline cases whose mutational status could be recalculated into 10 MM and 5 UM. The rest 7 remaining in borderline group. - We could identify both clones in 29 double rearrangements cases, with concordant mutational status except 2/29 undetermined cases, included in UM group regarding HTS results. Our tool led to the identification of a dominant clonotypic IgHV in all cases, and when compared the HTS sequence/mutational status for the most abundant clone with Sseq and for the IgHV status determination, 15 out of 22 (68,18%), could be reclassified. This case showed a major clone with productive rearrangement mutated by Sseq but unmutated by HTS. Conclusions: Analyze and determine the mutational status of the IgHV locus by HTS, would potentially reveal multiple rearrangements and increase the prognostic precision of IgHV mutation analysis. IgHV-HTS classification is able to precisely classify patients with borderline status or/and multiple IgHV rearrangements for which Sseq is inconclusive. In this case, it has been possible to improved prognostication for 17 out of 24 patients. This is helping us to discover the advantages of the data obtained by HTS compared with current Sseq standard technique. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Terol: Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Gilead: Research Funding; Roche: Consultancy; Astra Zeneca: Consultancy.

scholarly journals High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 986
Author(s):  
Saengchoowong ◽  
Khongnomnan ◽  
Poomipak ◽  
Praianantathavorn ◽  
Poovorawan ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Mdck Cells ◽  
Computational Prediction ◽  
Influenza B ◽  
Madin Darby Canine Kidney ◽  
High Throughput Analysis ◽  
Darby Canine Kidney ◽  
Canine Kidney

Victoria and Yamagata lineages of influenza B viruses are globally circulating in seasonal epidemics. Madin–Darby canine kidney (MDCK) cells are permissive for viral isolation and vaccine manufacture. Nevertheless, the interplay between influenza B viruses and host microRNAs has not been investigated in this cell line. Therefore, the present study aims at high-throughput analysis of canine microRNA profile upon infection of influenza B viruses. Briefly, MDCK cells were infected with Victoria or Yamagata lineage at MOI of 0.01. After being harvested at 6, 12 and 24 h post infection, microRNAs were subjected to high-throughput sequencing based on MiSeq platform (Illumina). The results demonstrated that five microRNAs including cfa-miR-197, cfa-miR-215, cfa-miR361, cfa-miR-1841, and cfa-miR-1842 were overexpressed in both Victoria and Yamagata lineage infections. Interestingly, computational prediction showed that karyopherin alpha 6 (KPNA6) was targeted by cfa-miR-197 and cfa-miR-215. Moreover, the binding sites of both microRNAs were assessed by 3′-UTR reporter assay. The results showed that only cfa-miR-197 could bind to the target sites of KPNA6, leading to suppressing luciferase activity. Additionally, silencing of KPNA6 was confirmed by overexpression of cfa-miR-197. This study provides canine microRNA responses to seasonal influenza B viruses, suggesting that virus-mediated microRNAs might play crucial roles in host gene regulation.

scholarly journals High-throughput sequencing analysis of genes encoding the B-lymphocyte receptor heavy-chain CDR3 in renal and peripheral blood of IgA nephropathy

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Dapeng Chen ◽  
Zheng Zhang ◽  
Yue Yang ◽  
Quan Hong ◽  
Wenge Li ◽  
...  
Keyword(s):  
Iga Nephropathy ◽  
High Throughput ◽  
Heavy Chain ◽  
Peripheral Blood ◽  
High Throughput Sequencing ◽  
Kidney Tissue ◽  
Renal Tissue ◽  
Chronic Glomerulonephritis ◽  
Sequencing Analysis ◽  
Variant Frequency

Abstract Aim: IgA nephropathy (IgAN) is one of the most common chronic glomerulonephritis. Its etiology and pathogenesis remain unclear. We thus explored the immune repertoire of the B-cell receptor (BCR) and the heavy-chain complementarity-determining region 3 (CDR3) in renal tissue and peripheral blood of IgAN patients. Method: Total RNAs extracted from renal tissues and peripheral blood of patients and peripheral blood of healthy controls (HCs) were analyzed via high-throughput multiplex PCR sequencing. We amplified and sequenced BCR heavy-chain CDR3 regions to explore repertoire diversity, V/J gene family distribution, CDR3 lengths, BCR heavy-chain variants, consistency between tissue and peripheral blood data, and clones ‘shared’ by these bodily compartments. Results: We identified the renal tissue and peripheral blood BCR heavy-chain CDR3 immune repertoires of 15 IgAN patients. Top1 could be more readily cloned from peripheral blood of patients than from controls (P<0.05), the average CDR3 length was significantly shorter in patients than in HCs (P<0.05), the variant frequency of the gene encoding the BCR heavy chain was higher in patients than in HCs (P<0.05), and the BCR variant frequency was highest in IgAN kidney tissue. Preliminary screening for ‘shared’ clones showed that, in at least 13 patients, the ‘ALYFHNSAY’, ‘ARWGPMYYYMDV’, ‘ARDQGALNA’, and ‘ARVDNPADF’ CDR3 sequences were evident in peripheral blood samples from patients, but not HCs. Conclusions: We found that the ‘ALYFHNSAY’, ‘ARWGPMYYYMDV’, ‘ARDQGALNA’, and ‘ARVDNPADF’ clonal sequences may be useful for noninvasive diagnosis and treatment planning in IgAN.

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